Myoglobin, refinement

Nilsson K et al (2004) The protonation status of compound II in myoglobin, studied by a combination of experimental data and quantum chemical calculations quantum refinement. Biophys J 87(5) 3437-3447... 

One simple case of disordered structure involves many of the long charged side chains exposed to solvent, particularly lysines. For example, 16 of the 19 lysines in myoglobin are listed as uncertain past C8 and 5 of them for all atoms past C/J (Watson, 1969) for ribonuclease S Wyckoff et al. (1970) report 6 of the 10 lysine side chains in zero electron density in trypsin the ends of 9 of the 13 lysines refined to the maximum allowed temperature factor of 40 (R. Stroud and J. Chambers, personal communication) and in rubredoxin refined at 1.2 A resolution the average temperature factor for the last 4 atoms in the side chain is 9.2 for one of the four lysines versus 43.6, 74.4, and 79.3 for the others. Figure 57 shows the refined electron density for the well-ordered lysine and for the best of the disordered ones in ru-... 

Finally, we note that low-temperature crystallographic studies have been carried out on one nucleic acid, the 5-DNA dodecamer whose room-temperature structure was solved in Dickerson s laboratory (Dickerson, 1981). Refinement at 16 K revealed a large overall drop in B, but some of the atoms in the molecule still had very large B-factors even at this very low temperature. These large residual mean-square displacements were interpreted as demonstrating the presence of static disorder however, by analogy with the results on myoglobin, a disorder which is dynamic at room temperature but becomes frozen into a static distribution at low temperature is also consistent with the observations. It is also possible that the disorder in these atoms is dynamic even at 16 K this point has been considered by Hartmann et al. (1982). 

Since the dispersive properties of helices in what will be taken as the standard sense, now known to be right-handed as in myoglobin, have been the most thoroughly studied and since a case can be made that it is the predominant sense in proteins, this review will focus on the capacity of optical rotatory methods to discern mixtures of this conformation with disordered regions. It will discuss the manner in which theoretical considerations have provided the forms into which rotatory data are currently cast, the calibration of their constants by studies of synthetic polypeptides in known conformation, and then the application of these equations and scales in the structural interpretation of the rotatory dispersion of proteins. This pattern of analysis will undoubtedly undergo refinement and revision as these methods are applied to new species of polypeptide and protein in concert with other means of conformational assignment. In particular, an extension of the spectral range of measurement toward optically active absorption bands in the far ultraviolet can be expected to yield new information about the rotatory power of the peptide bond and thus enhance the interaction of theory and observation that has already proved fruitful. 

Cheng, X., and Schoenborn, B. P. Repulsive restraints for hydrogen bonding in least-squares refinement of protein crystals. A neutron diffraction study of myoglobin crystals. Acta Cryst. A47, 314-317 (1991). 

Since we submitted this chapter we have slightly refined our calculation of thermal transport coefficients for myoglobin and green fluorescent protein [163], as well as for water [164]. 

For those protein models for which structure amplitudes have been calculated, R values are usually in the range 0.4-0.5 even for the limited data available for these noncentrosymmetric structures.1S 18 There would appear to be no in principle reason why a protein model should not refine within the limits imposed by the extent and accuracy of the data and, in fact, efforts were made to refine myoglobin, the first protein to be solved. Considerable effort was invested and progress was made, but the work has never been fully reported. It is clear from what has been reported, however, that the effort was an extensive one but that the methods used were not sufficiently effective. 

Of interest also are the results concerning deviations of the atomic fluctuations from simple isotropic and harmonic motion. As discussed in Chapt. XI, most X-ray refinements of proteins assume (out of necessity, because of the limited data set) that the motions are isotropic and harmonic. Simulations have shown that the fluctuations of protein atoms are highly anisotropic and for some atoms, strongly anharmonic. The anisotropy and anharmonicity of the atomic distribution functions in molecular dynamics simulations of proteins have been studied in considerable detail.193"197 To illustrate these aspects of the motions, we present some results for lysozyme196 and myoglobin.197 If Ux, Uy, and Uz are the fluctuations from the mean positions along the principal X, Y, and Z axes for the motion of a given atom and the mean-square fluctuations are... 

Figure 61. Positional error in refinement of the 25-ps data from myoglobin simulation The deviations between atomic positions are calculated after the molecular dynamics average structure and the refined structures are superimposed by least squares. The deviations for backbone atoms (N, C, and C", solid line) and sidechain atoms (dotted line) are averaged over residues.

Figure 62, Residue averages of mean-square fluctuations from molecular dynamics (dotted line) and refinements (solid line). All plots are for the results of refining the 25-ps data from myoglobin simulation, (a) Backbone (N, C, and C ) averages (b) sidechain averages.

Figure 63. Scatter plots or mean-square fluctuations calculated from the myoglobin simulation and from the refinements. All the atoms are included in these plots. The exact mean-square fluctuations, , calculated directly from the simulations, are plotted along the Y axis. The refined mean-square fluctuations, obtained from the refined temperature factors, are plotted along the X axis, (a) Results of a restrained refinement of the 25-ps data (b) results from a refinement of the 300-ps simulation data with loose restraints.

In contrast to the compressibility, the thermal expansion of proteins has received much less attention. Frauenfelder et al. [65] have estimated the thermal expansion of myoglobin from the refined X-ray structure at 80 and 255-300K. They conclude that the expansion comes mainly from the subatomic free volumes between the atoms. The expansion obtained from the temperature dependence of the vibrational frequency shifts of the hydrogen bonds support... 

The neutron diffraction studies on myoglobin by Benno Schoenborn, reported elsewhere in this symposium, sound an important note of warning. Whereas in 1971 he had reported 106 water molecules per myoglobin molecule by neutron diffraction (33) and Takano in refined x-ray diffraction studies had found about 80 (34), Schoenborn in very careful work has now reduced his estimate to 42. It is known that false peaks of density can often be produced in calculations from x-ray or neutron diffraction. How many alleged water molecules in other structures may there be, that will not withstand closer scrutiny in future This is a disturbing question that can only be answered by further research. 

Results from an early unrefined met myoglobin neutron analysis ( ) based on the x ray structure by Watson and Kendrew ( ) showed a total of 106 water peaks. In that analysis, only peaks that had a peak height equivalent to one-half D2O molecule were considered. More recently, Takano (O, in a refined high resolution x-ray analysis of met myoglobin, found 72 water molecules, and in the present refined neutron analysis of CO-Mb, only AO water molecules were found. These water molecules were identified in a difference map where the features depicting the protein had been subtracted. The protein structure itself had been refined by successive real-space refinement of eight... 

Inspection and analysis of the Mb-CO difference map resulted in a finding of 40 water molecules per asymmetric unit (one myoglobin molecule). No peak with a weight of less than 0.4 water molecule was included in this number. Several features in the remaining difference map might represent water molecules, but at a low level of occupancy (<0.4). Refinement of the water locations and weight was performed by two cycles of real-space... 

A further refinement to the production of heme protein models was the synthesis of doubly-strapped models containing different straps. As models for hemoglobin or myoglobin, incorporation of a nitrogen base into one strap would simulate the proximal face of the natural system while the steric encumbrance provided by the second strap would mimic the distal, oxygen-binding face. 

The computer also played an enormously important part in protein crystallography at Cambridge. The elucidation ot the structure of horse myoglobin by Kendrew and his co-workers, announced in 1958, would not have been possible without EDSAC on which to process the 400 reflections measured. Subsequently EDSAC2 was used to produce a more refined structure based on 10,000 reflections and also to produce a determination of the structure of haemoglobin by Perutz and his co-workers. Both of these achievements were reported in Nature in... 

The vast majority of fast ligand substitutions studied to date are in the s, ms, and fjLS time scales, which encompass a vast range of reactions, and for which fast reaction techniques have become both refined and readily commercially available. Not surprisingly the majority of reactions reported in this chapter fall into these time scales, and have been studied predominantly by stopped-flow, temperature-jump, or nuclear magnetic resonance fast reaction techniques, which are referred to by the initials SF, TJ, and NMR hereafter. However, substantial rewards await those who venture into the ns and ps time scale as shown by a study of the recombination kinetics of small ligands at the Fe(II) center of sperm whale and elephant myoglobins in which laser pulses of 1 ps and 4 ns facilitated the determination of rate constants in the range 3 x 10 to 5 x s and 10 to 10 ... 

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